Virus Surface Antigen Gene Research Articles

A one-tube multiplexed colorimetric strategy based on plasmonic nanoparticles combined with non-negative matrix factorization

Herein, a one-tube colorimetric platform has been developed for the simultaneous determination of two analytes (DNA as model object) in one tube with picomolar sensitivity. SPR-active nanoparticles are used to encode reporter probes sensitive to oligonucleotides associated with hepatitis A virus Vall7 polyprotein gene (HVA) and hepatitis B virus surface-antigen gene (HVB) respectively and magnetic beads (MBs) serve as the removal tool. In this mixed nanoparticles based biosensor, the addition of target analytes could change the concentration of each nanoparticle, leading to different colors of the supernatant. The influence of spectral overlap has been eliminated by a non-negative matrix factorization (NMF). With the assistance of NMF, the limit of detection (LOD) can be determinated as pM level without amplification. On the whole, this nanosensor boasts the advantages of high sensitivity and low sample consumption. Simultaneous colorimetric detection and quantification of two molecules in one tube are demonstrated.

High doses of siRNAs induce eri-1 and adar-1 gene expression and reduce the efficiency of RNA interference in the mouse

RNAi (RNA interference) is a gene-silencing mechanism that is conserved in evolution from worm to human and has been a powerful tool for gene functional research. It has been clear that the RNAi effect triggered by endogenous or exogenous siRNAs (small interfering RNAs) is transient and dose-dependent. However, there is little information on the regulation of RNAi. Recently, some proteins that regulate the RNA-silencing machinery have been identified. We have observed in previous work that the expression of target genes rebounds after being suppressed for a period of time by siRNAs. In the present study, we used secretory hepatitis B virus surface antigen gene as a reporter and compared its expression level in cell culture and mice challenged by different doses of siRNAs. A quicker and higher rebound of gene expression was observed in mice tail-vein-injected with higher doses of siRNA, and the rebound was associated with an increase in the mRNA level of meri-1 (mouse enhanced RNAi) and adar-1 (adenosine deaminase acting on RNA) genes encoding an exonuclease and RNA-specific adenosine deaminase respectively. Down-regulation of meri-1 by RNAi enhanced the sensitivity and efficiency of siRNA in inhibiting the expression of hepatitis B virus surface antigen. These results indicate that RNAi machinery may be under negative regulation, through the induction of a series of genes coding for destabilizing enzymes, by siRNAs introduced into the cell, and also suggest that a suitable amount of siRNA should be used for research or therapeutic applications.

Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells

We have examined the antiviral activity of the crude extract prepared from the root of Saussurea lappa Clarks, a Chinese medicinal herb which is widely used for many illneses including cancer. Two active components, costunolide and dehydrocostus lactone, were identified which show strong suppressive effect on the expression of the hepatitis B surface antigen (HBsAg) in human hepatoma Hep3B cells, but have little effect on the viability of the cells. Both costunolide and dehydrocostus lactone suppress the HBsAg production by Hep3B cells in a dose-dependent manner with IC 50s of 1.0 and 2.0 μM, respectively. Northern blotting analysis shows that the suppression of HBsAg gene expression by both costunolide and dehydrocostus lactone were mainly at the mRNA level. Furthermore, the suppressive effect of costunolide and dehydrocostus lactone on HBsAg and hepatitis B e antigen (HBeAg), a marker for hepatitis B viral genome replication in human liver cells, was also observed in another human hepatoma cell line HepA 2 which was derived from HepG 2 cells by transfecting a tandemly repeat hepatitis B virus (HBV) DNA. Similarly, the mRNA of HBsAg in HepA 2 cells was also suppressed by these two compounds. Our findings suggest that costunolide and dehydrocostus lactone may have potential to develop as specific anti-HBV drugs in the future.

Inhibition of hepatitis B virus surface antigen gene expression in carcinogen-induced liver tumors from transgenic mice.

We previously showed that hepatitis B surface antigen (HBsAg)-producing transgenic mice were more sensitive to hepatocarcinogens than their normal littermates were. We have now investigated the regulation of hepatitis B virus (HBV) gene expression in carcinogen-induced liver tumors of HBV-carrier transgenic mice and in three cell lines derived from tumor samples. Transcription of the S gene was repressed in 17 tumors even though they had normal levels of liver-specific mRNAs such as albumin and transferrin. Three hepatoma cell lines, derived from independent tumor samples, were analyzed for their capacity to express the S gene after transfection of cloned DNA. Although they no longer expressed the endogenous S gene, they were still able to express it from transfected viral DNA both transiently and stably. The loss of HBsAg expression in tumors and in the cell lines was accompanied by de novo methylation of the S region, which is a way to permanently repress gene expression. Our data confirm in an animal model previous observations of S-gene expression in human hepatocarcinoma and suggest a role for its downregulation in tumor progression.

Modulation of expression of the hepatitis B virus surface antigen gene by the viralX-gene product

Human hepatoma cells (HepG2 and HUH7) transfected with a plasmid (pHBV1004) containing the transcription units for the major surface antigen (S) mRNA and the X mRNA of hepatitis B virus (HBV) secreted surface antigen (HBsAg) into the culture medium. A frameshift mutation in the X gene carried by pHBV1004 greatly reduced HBsAg production by cells transfected with an equivalent amount of the mutant (pHBV1004-B). The mutation could be complemented by cotransfection with a plasmid (pSV2HBX) containing the X structural gene under control of the SV40 early promoter. HBsAg production by cells cotransfected with pHBV1004-B and pSV2HBX was equivalent to that in cells transfected with the parent plasmid (pHBV1004) alone. Levels of HBsAg production were directly related to the amount of S mRNA produced, showing that the X-gene product (HBxAg) can modulate expression of the S gene.

Cattle leukaemia vaccine containing recombinant vaccinia virus incorporating cattle leukaemia virus surface antigen gene

Cattle leukaemia vaccine containing recombinant vaccinia virus incorporating cattle leukaemia virus surface antigen gene

Involvement of an AFP1-binding site in cell-specific transcription of the pre-S1 region of the human hepatitis B virus surface antigen gene

Human hepatitis B virus infection is characterized by a high degree of hepatotropism which may be due to the dependency of viral genes on specific host factors for their expression. To learn more about such a requirement and the molecular basis of the viral tissue tropism we analyzed the promoter function in the pre-S1 region of the surface antigen gene. DNase I footprinting and competition gel retardation assays showed that a sequence with an AT-rich core (AT motif) in the pre-S1 promoter region interacts with AFP1, a hepatoma nuclear factor that binds to the alpha-fetoprotein enhancer and promoter. Functional analysis of the pre-S1 AT motif by transient transfection assays showed that this element is important in cell-specific transcriptional initiation. These results suggest that AFP1 may be one of the factors determining the liver specificity of human hepatitis B virus.

The hepatitis B virus enhancer modulates transcription of the hepatitis B virus surface antigen gene from an internal location.

We have used a transient expression system to examine the influence of the hepatitis B virus enhancer on the transcription of the hepatitis B virus surface antigen (HBsAg) gene. This enhancer is located 3' to HBsAg coding sequences but is contained within the mature HBsAg transcripts. Removal of the enhancer resulted in a significant reduction in HBsAg gene expression in liver cells. By using an in vitro nuclear run-on assay, this effect was determined to be at the level of transcription. We have further noted that HBsAg expression can be restored by the reinsertion of the enhancer sequences in the enhancerless vector. However, this reconstitution of HBsAg expression appears to depend on location of the enhancer with regard to the HBsAg promoters.

Recombinant adenovirus induces antibody response to hepatitis B virus surface antigen in hamsters.

Recombinant adenoviruses carrying the hepatitis B virus surface antigen coding sequence in the adenovirus E3 region were constructed using DNA from either adenovirus type 5 or an adenovirus type 5 E3-region deletion mutant. Both of these recombinant adenoviruses replicated as efficiently as wild-type adenovirus in all human cells tested, including the human diploid cell strain WI-38. This indicates that insertion of the hepatitis B virus surface antigen gene into the E3 region does not significantly affect viral replication. Human cells infected with these recombinant adenoviruses secreted immunoreactive hepatitis B virus surface antigen. Since a practical small animal model for human adenoviruses was lacking, a hamster model was developed to evaluate the immunogenic potential of these recombinant adenoviruses. Upon intranasal inoculation, both wild-type adenovirus and the adenovirus E3-region deletion mutant replicated in the lungs of these animals and induced an antibody response against adenovirus. Hamsters similarly immunized with the live recombinant adenoviruses produced antibody against both adenovirus and hepatitis B virus surface antigen.

Stability of the artificial chimeric plasmid in yeast

The Hepatitis B virus surface antigen gene was cloned into a yeast plasmid vector with a complete 2-micron origin and an ars origin, and the resultant recombinant plasmid, pMYN101, was transformed into several yeast strains, AH22, AH22 cir-0, A281, and LL20 cir-0. The recombinant plasmid was found to be very stable in all of these strains tested suggesting that the phenomenon of plasmid incompatibility due to the coexistence with another 2-micron plasmid does nol occur. For comparison a few other kinds of yeast plasmids with an incomplete 2-micron origin or an ars orgin were transformed into the same yeast hosts and their stability was sludied in a batchwise operation. These plasmids showed an unstable yet interesting behaviour making careful prediction of plasmid stability based upon the complete characteristics of both the plasmids and the hosts, and their interaction an important necessity.

Increased expression of hepatitis B virus surface antigen gene in Escherichia coli by IS1 insertion

We selected faster growing colonies of Escherichia coli harbouring an expression plasmid for hepatitis B virus surface antigen (HBsAg) gene after mutagenesis. Among these colonies, three were found to produce an increased level of HBsAg as a consequence of alteration of the plasmid. Analysis of this plasmid showed that an insertion sequence, IS1, was inserted into a middle region of the HBsAg gene (codon for Pro 127) to generate a termination codon 20 bp downstream from the junction site between the HBsAg gene and the left end of IS1. Insertion of a chemically synthesized termination codon into the same region of the HBsAg gene also increased the expression of the HBsAg gene. These results suggest that HBsAg lacking the COOH-terminal region is produced at a high level because it does not inhibit the growth of the host.

Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene

Using dodecadeoxynucleotides as primers for DNA synthesis and 3 '- o-chlorophenyl-phosphorylated dodecadeoxynucleotides as “stoppers” for chain elongation, pre-defmed regions of a gene previously cloned in M 13 single-stranded (ss) DNA phage were converted into double-stranded (ds) DNA utilizing the action of the Klenow fragment of Escherichia coli DNA polymerase I (Pollk). The resulting ds DNA was freed from the ss region by S 1 nuclease treatment. This method can be used to obtain DNA fragments of any size with pre-defined 5' and 3' ends. About 15% of the input ss DNA template molecules are converted into ds DNA fragments. This technique was used to synthesize several DNA fragments from different portions of the hepatitis B virus surface antigen (HBsAg) gene. The products were then ligated into a yeast plasmid vector that carries the E. coli lacZ gene which is located downstream from the yeast acid-phosphatase promotor. Using this system, several fragments of HBsAg were produced in the form of β-galactosidase fused protein.

The transient expression of genes following DNA transfection: Dependence on the cell line

Summary In order to study why some cells, even those poorly susceptible to stable biochemical transformation such as many primate cells, transiently express a gene at a high level during the first days after transfection, we compared the monkey kidney Vero and mouse L cells for plasmid penetration and persistence, as well as for gene transcription after transfection. We report here that plasmid penetration and persistence were comparable in both types of cells, and about 500 plasmid molecules were found per cell one week after transfection. This amount decreased thereafter, but free plasmid molecules were detected for at least 3 weeks in Vero as well as in L cells. In contrast, a difference existed between the two cell lines at the transcriptional level: the transcription efficiency of the transfected genes—or mRNA stability—was higher in Vero than in mouse L cells. Similar results were obtained with two genes, the human hepatitis B virus surface antigen gene and the aminoglycoside-3′-phosphotransferase gene, indicating that the high transient expression was not specific for the virus antigen gene in Vero cells, but depended on the cell line.

HBV DNA and applications - hepatitis B virus surface antigen gene cloning and expression in animal cell culture for vaccine preparation

HBV DNA and applications - hepatitis B virus surface antigen gene cloning and expression in animal cell culture for vaccine preparation

Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids.

We examined the transcription of the hepatitis B virus surface antigen (HBsAg) gene in COS cells transfected with simian virus 40-based recombinant plasmids. When positioned behind the simian virus 40 late promoter, three transcripts were identified which hybridized to the HBsAg gene: a 2,000-nucleotide transcript colinear with a gene, a 1,100-nucleotide transcript representing a spliced molecule in which a major portion of the sequences encoding HBsAg were deleted, and an 800-nucleotide transcript derived primarily from sequences 3' to the HBsAg gene. The splice acceptor site utilized by the 1,100-nucleotide transcript is located immediately upstream of an open reading frame of unknown function contained within the 3' nontranslated region of the HBsAg gene. The HBsAg-specific mRNA species terminate 12 to 19 base pairs 3' of the sequence UAUAAA, similar to the concensus hexanucleotide which is thought to promote polyadenylation (AAUAAA). We constructed a series of plasmids with progressive deletions from the region surrounding where these transcripts terminate. Analysis of mRNA produced by cells transfected with these plasmids indicated that the signal hexanucleotide is in itself unable to promote the efficient processing of mRNA in the absence of downstream hepatitis B virus sequences. Processing proceeds properly, however, from plasmids containing an additional 30 nucleotides 3' of this signal.

Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids.

We examined the transcription of the hepatitis B virus surface antigen (HBsAg) gene in COS cells transfected with simian virus 40-based recombinant plasmids. When positioned behind the simian virus 40 late promoter, three transcripts were identified which hybridized to the HBsAg gene: a 2,000-nucleotide transcript colinear with a gene, a 1,100-nucleotide transcript representing a spliced molecule in which a major portion of the sequences encoding HBsAg were deleted, and an 800-nucleotide transcript derived primarily from sequences 3' to the HBsAg gene. The splice acceptor site utilized by the 1,100-nucleotide transcript is located immediately upstream of an open reading frame of unknown function contained within the 3' nontranslated region of the HBsAg gene. The HBsAg-specific mRNA species terminate 12 to 19 base pairs 3' of the sequence UAUAAA, similar to the concensus hexanucleotide which is thought to promote polyadenylation (AAUAAA). We constructed a series of plasmids with progressive deletions from the region surrounding where these transcripts terminate. Analysis of mRNA produced by cells transfected with these plasmids indicated that the signal hexanucleotide is in itself unable to promote the efficient processing of mRNA in the absence of downstream hepatitis B virus sequences. Processing proceeds properly, however, from plasmids containing an additional 30 nucleotides 3' of this signal.

Expression of hepatitis B virus surface antigen gene in cultured cells by using recombinant plasmid vectors.

By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.

Expression of the hepatitis B virus surface antigen gene in cell culture by using a simian virus 40 vector.

We have constructed a simian virus 40 recombinant carrying a fragment of DNA from hepatitis B virus. Cultured monkey kidney cells infected with this recombinant produce hepatitis B surface antigen. The antigen is excreted into the culture medium as 22-nm particles with the same physical properties, antigenic composition, and constituent polypeptides as those found in the sera of patients with type B hepatitis.